Synthesis and antinociceptive activity of chimonanthines and pyrrolidinoindoline-type alkaloids

Article abstract of DOI:10.1016/S0968-0896(02)00078-0

Hodgkinsine, a trimeric pyrrolidinoindoline type alkaloid, present as a major constituent of Psychotria spp. (Rubiaceae), has shown to produce dose-dependent, naloxone reversible, analgesic effect in thermal models of nociception and in the capsaicin-induced pain. SAR studies have been initiated by synthesizing the three diastereomeric dimers (chimonanthines) (11-13) which were evaluated in vitro and in vivo along with the synthetic intermediates. Strong binding affinities for μ opioid receptors were found for (-)- and (+)-chimonanthine monourethanes (9 and 10), whereas (-)-, (+)- and (meso)-chimonanthine (11-13) and hodgkinsine displayed low affinity. In vivo data have shown that only (+)-chimonanthine (12) and calycosidine resemble the analgesic profile found for hodgkinsine.

Full text of DOI:10.1016/S0968-0896(02)00078-0

Bioorganic & Medicinal Chemistry 10 (2002) 2133–2142
Synthesis and Antinociceptive Activity of Chimonanthines and
Pyrrolidinoindoline-Type Alkaloids
L. Verotta,a,* F. Orsini,a M. Sbacchi,b M.A. Scheildler,b T.A. Amadorc
and E. Elisabetskyc,d
a
`
Dipartimento di Chimica Organica e Industriale, Universita degli Studi di Milano,via Venezian 21, 20133 Milan, Italy
bNeurobiology Research, GlaxoSmithKline, Via Zambeletti, I-20021 Baranzate di Bollate, Milan, Italy
c
´
Curso de Pos-graduac¸a˜o em Ciencias Biologicas-Bioquımica, Universidade Federal do Rio Grande do Sul,
´
ˆ
´
Ramiro Barcelos 2600/anexo, Porto Alegre/RS, Brazil
dDepartamento de Farmacologia, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite 500/202,
90040-100, Porto Alegre/RS, Brazil
Received 2 January 2002; accepted 12 February 2002
Abstract—Hodgkinsine, a trimeric pyrrolidinoindoline type alkaloid, present as a major constituent of Psychotria spp. (Rubiaceae),
has shown to produce dose-dependent, naloxone reversible, analgesic effect in thermal models of nociception and in the capsaicin-
induced pain. SAR studies have been initiated by synthesizing the three diastereomeric dimers (chimonanthines) (11–13) which were
evaluated in vitro and in vivo along with the synthetic intermediates. Strong binding affinities for m opioid receptors were found for
(ꢀ)- and (+)-chimonanthine monourethanes (9 and 10), whereas (ꢀ)-, (+)- and (meso)-chimonanthine (11–13) and hodgkinsine
displayed low affinity. In vivo data have shown that only (+)-chimonanthine (12) and calycosidine resemble the analgesic profile
found for hodgkinsine. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
of at least three types of opioid receptors, namely m, k
and d, has been well established.3 Specific types of
receptors mediate the pharmacological actions of
opioids, either therapeutic or euphorigenic, as well as
the reinforcing properties which may lead to addiction.
Therefore, the detection of opioid agonists that would
maintain the analgesic profile of morphine, while lack-
ing its addictive properties, is highly desirable.
New analgesics have been constantly and intensively
sought by the pharmaceutical industry.1 This enduring
interest is related to the still significant limitations (e.g.,
side effects, effectiveness, tolerance and dependence) of
presently available compounds. Despite of the remark-
able efficacy of morphine and other opiates, chronic
pain, perhaps more diffused than acute pain, cannot be
effectively treated with opiates. Furthermore, neuro-
pathic pain following injury to the nervous system
responds poorly to opiates at doses that do not cause
serious adverse effects. Thus, alternative management
strategies need to be considered for dealing with situa-
tions where pain is unresponsive to currently available
medication.
The increasing recognition of the vast chemical diversity
in tropical forests,4 associated with the development of
revolutionary techniques for isolation and elucidation
of chemical compounds and characterization of their
pharmacological properties, justify the rekindled inter-
est in natural products.5,6,7 Surveys of medicinal plants
used among Amazonian caboclos (rural peasants) of the
State of Para (Brazil) pointed to Psychotria colorata
(Will. ex R. & S.) Muell. Arg., traditionally used for
‘treatment of earache’ and ‘calming abdominal pain’.8
We further reported that alkaloids present in leaves and
flowers of P. colorata have marked analgesic activity, as
evaluated through various pain models.9,10,11 Phyto-
chemical analyses of P. colorata flowers identified oli-
gomeric pyrrolidinoindoline alkaloids as major
The physiological modulation of pain involves opioid
receptors, exhibiting a widespread distribution in the
central and peripheral nervous systems.2 The existence
*Corresponding author. Tel.: +39-02-5031-4114; fax: +39-02-5031-
4106; e-mail: luisella.verotta@unimi.it
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00078-0

2134
L. Verotta et al. / Bioorg. Med. Chem. 10 (2002) 2133–2142
Scheme 1. (i) CIOOCH3, CH2Cl2/Et3N, rt: (ii) TTFA/ethereal BF3, rt; (iii) (ꢀ)-(S)-(a-methyl-benzyl)isocyanate, reflux; (iv) LiAlH4/THF, reflux;
(v) C5H11OH/C5H11ONa, reflux.
components,11,12 these alkaloids seem to be specific to
Psychotria sps, and closely related genus.
have studied.13 Because these compounds are present in
the extracts in low concentrations, it is expected that
they do not significantly contribute to the overall
activity of the traditional plant-based preparations.
Nevertheless, a sustainable supply of all the three chi-
monanthines could represent a valid starting point for
the structure–activity studies.15
The trimer hodgkinsine is one of the most abundant
pyrrolidinoindoline alkaloids present in extracts of Psy-
chotria species collected in different places and sea-
sons.13 Pharmacological analyses revealed that
hodgkinsine produces
a dose-dependent, naloxone
reversible, analgesic effect in thermal models of noci-
ception, suggesting that activation of opioid receptors
participates in its mode of action. Hodgkinsine is like-
wise a potent dose-dependent analgesic in the capsaicin-
induced pain, suggesting that the glutamate nmda
receptors are also likely to be involved.14
The purpose of this study is to generate preliminary
data relevant to the analgesic activity of chimonanthines
and the intermediates obtained during the synthetic
pathway in choosen models of analgesia.
Synthesis
The synthesis of hodgkinsine is complicated by the
number of existing stereocenters. A number of other
trimers are present in the extracts,13 nevertheless, the
difficulties in defining their absolute configurations limit
a rational approach to a possible drug development.
The oxidative dimerization of carbomethoxytryptamine
(2) with thallium trifluoroacetate (TTFA) allowed to
obtain (ꢁ)-carbomethoxychimonanthine (3) and
(meso)-carbomethoxychimonanthine (4). The (meso)-
derivative was directly reduced with LiAlH4 to give
(meso)-chimonanthine (13).
The first members of the oligomeric pyrrolinoindoline
alkaloids series, namely the three diastereomeric dimers,
(+)-, (ꢀ)-, and (meso)-chimonanthine can be easily
prepared following the protocol here described (Scheme
1). From these only (+)- and (meso)-chimonanthine are
found in the alkaloid extracts of the Psychotria sps we
The racemate (3) was resolved by treatment with (ꢀ)-
(S)-(a-methyl-benzyl) isocyanate. Mono-urethanes of
(ꢀ)- and (+)-carbomethoxychimonanthine (5–6) were
obtained, accompanied by a 20% of the corresponding

L. Verotta et al. / Bioorg. Med. Chem. 10 (2002) 2133–2142
2135
diurethanes (7–8). Monourethanes of carbomethox-
ychimonanthines (5) and (6) were separately reduced
with LiAlH4 to give monourethanes of (ꢀ)- and (+)-
chimonanthine (9)-(10) along with a 20% of, respec-
tively, (ꢀ)- and (+)-chimonanthine (11–12). (ꢀ)- and
(+)-chimonanthine were finally prepared by alkaline
hydrolysis of the corresponding monourethanes (9–10)
(see Scheme 1).
derivatives led us to prepare the diastereomeric carba-
mates (5–6).
Results and Discussion
Chimonanthines and their precursors, obtained through
the synthetic pathway shown in Scheme 1, were tested
on m- and k-opioid binding assay, and on the tail-flick
and the capsaicin-induced pain models.
In an alternative pathway, a first reduction of 3, fol-
lowed by racemate resolution through (ꢀ)-(S)-(a-
methyl-benzyl) isocyanate led to lower yields, probably
due to the formation of N1-formyl and N1-hydroxy-
methylene derivatives as by products, which were not
obtained when a N8-protecting group is present.
Opioid binding
The binding affinities of chimonantines and pyrrolidino-
indoline-like analogues for the m- and k-opioid receptors
are reported in Table 1. Strong binding affinities (low
nanomolar range) towards m receptor were found for
both (+)- and (ꢀ)-chimonanthine monourethanes (9)
and (10), whereas (ꢀ)-chimonanthine (11), meso-chi-
monanthine (13) and hodgkinsine displayed much lower
affinities to m receptor than morphine or the mono-
urethanes. Even lower affinity was found for (+)-chimo-
nanthine (12), whereas no affinity for carbo-
methoxychimonanthine (4) could be detected.
The oxidative dimerization of carbomethoxy-
tryptamine16 is the limiting factor for obtaining high
yields from the reported sequence of reactions, but it
easily allowed preparation in one step of the three chi-
monanthines. The reaction is sensitive to the N1 acyl
derivative; in fact, the same protocol applied to the
product obtained from tryptamine and menthyl-
chloroformiate was completely unsuccessful.
Initially, the racemate resolution was tentatively
approached through the preparation of (+)-O,O0-
dibenzoyl-d-tartrates, but the low yields of crystalline
All these compounds, tested in a selective k-opioid
binding assay, showed poor affinities with Ki values in
the micromolar range.
Figure 1. Effects of morphine (M), chimonanthines, hodgkinsine and calycosidine on the tail flick test. 1a dose–effect analysis of the (+)- and (ꢀ)-
chimonanthine (12) and (11); 1b effects of (+)- and (ꢀ)-chimonantine monourethanes (10) and (9); 1c effects of rac-chimonanthine, (meso)-carbo-
methoxychimonanthine (4) and meso-chimonanthine (13); 1d effects of hodgkinsine and calycosidine. N=6–8. S=saline. Columns represent % of
maximum possible effect (%MPE) and vertical bars SEM. *=p<0.05; **=p<0.01, ANOVA/Student–Newman–Keuls.

2136
L. Verotta et al. / Bioorg. Med. Chem. 10 (2002) 2133–2142
Tail-flick
(10 mg/kg). Figure 1b shows that monourethanes of
(ꢀ)- and (+)-chimonanthine (9 and 10) are equally
active at 1002 mmol/kg (5 mg/kg), 71 and 62% of MPE,
respectively, at the same dose. Figure 1c shows that
(rac)-(chimonanthine), (meso)-chimonanthine (13) and
(meso)-carbomethoxychimonanthine (4) do not bring
about significant effect in any of the doses tested. Figure
1d shows that hodgkinsine 965 mmol/kg (5 mg/kg)
results in 86% of MPE, comparable to morphine, and
calycosidine 1930 mmol/kg (10 mg/kg) results in 70% of
MPE.
Figure 1 shows results with the tail-flick model. (+)-
chimonanthine (12) presents 66% of MPE at 1445
mmol/kg (5 mg/kg) [compared to morphine 790 mmol/kg
(6 mg/kg)]) (Fig. 1a); nevertheless, it presents a bell
shaped dose–effect relationship with 44% of MPE at
2890 mmol/kg (10 mg/kg). Its enantiomer (ꢀ)-chimo-
nanthine (11) presents 40% of MPE at 2890 mmol/kg
Table 1. Receptor binding affinities of chimonanthines and pyrroli-
dinoindoline-type alkaloid analogues to the human m- and k-opioid
receptors
Capsaicin-induced pain
Compound
(m) Ki (nM)
(k) Ki (nM)
Figure 2 shows data from capsaicin-induced pain. Fig-
ure 2a shows that (+)-chimonanthine (12) results in
38% inhibition at 72.2 mmol/kg (0.25 mg/kg) and lacks
a clear dose–effect relationship. (ꢀ)-Chimonanthine (11)
72.2 mmol/kg (0.25 mg/kg) results in 47% inhibition of
licking time, and looses activity at 144.5 mmol/kg (0.5
mg/kg). Figure 2b shows that monourethanes of (+)-
and (ꢀ)-chimonanthine are inactive in the same mg/kg
dose range. Figure 2c shows that (rac)-chimonanthine at
144.5 mmol/kg (0.5 mg/kg) results in 29% inhibition
4
9
10
11
12
>2500
5.7ꢁ1.4
4.8ꢁ0.6
271ꢁ85
652ꢁ159
341ꢁ29
277ꢁ49
0.76ꢁ0.04
>2500
996ꢁ263
ꢂ2500
>2500
>2500
1447ꢁ45
13
Hodgkinsine
Morphine
>1000
63.9ꢁ10.5
Data are expressed as meanꢁSEM derived from at least three inde-
pendent determinations.
Figure 2. Effects of the chimonanthines, hodgkinsine and calycosidine in capsaicin-induced pain. 2a Dose–effect analysis of (+)-chimonanthine (12)
and (ꢀ)-chimonanthine (11); 2b effects of (+)- and (ꢀ)-chimonantine monourethanes (10) and (9); 2c effect of rac-chimonanthine, (meso)-carbo-
methoxychimonanthine (4) and meso-chimonanthine (13); 2d effects of hodgkinsine and calycosidine. N=6–10. S=saline. Columns represent per-
centage of inhibition and the vertical bars SEM. *=p<0.05, ANOVA/Student–Newman–Keuls.

L. Verotta et al. / Bioorg. Med. Chem. 10 (2002) 2133–2142
2137
[being inactive at 722.0 and 1445 mmol/kg (2.5 and 5.0
mg/kg)] that (meso)-carbomethoxychimonanthine (4) at
1125 mmol/kg (5 mg/kg) results in 45% inhibition of
licking time, and that (meso)-chimonanthine (13) at
144.5 and 722.0 mmol/kg (0.5 and 2.5 mg/kg) results in
30–35% inhibition, losing activity at 1445 mmol/kg (5.0
mg/kg). Hodgkinsine and calycosidine decreased dose-
dependently the licking time in the same dose range
(Fig. 2d).
tionship similar to that of morphine. Of the two basic
centers N1 and N8 present in the molecules here stud-
ied, the weak hydrogen acceptor N8 does not appear to
be particularly critic with respect to the activity, a fact
demonstrated by the strong activity of (ꢀ)- and (+)-
chimonanthine urethanes (9–10). On the contrary, N1 is
required to maintain the basic character, such as in
morphine, as shown by the lack of activity of carbo-
methoxychimonanthine (4).
It is important to note that the purpose of this pharma-
cological analysis was to differentiate active from inactive
compounds, comparing activities when possible.
A 3a(R) configuration of the pyrrolidinoindoline unit
seems to be favorable for maintaining activity, since
calycosidine, the product obtained by acid treatment of
hodgkinsine (see Experimental), is as active as hodg-
kinsine in the tail-flick model (see Fig. 1d). Since the bis-
quinoline part of the molecule is devoid of activity in
this model, as demonstrated by the inactivity of (ꢀ)-
calycanthine (data not shown), the presence of a pyrro-
lidinoindoline unit possessing a 3a(R) configuration can
be considered preferential.
A surprisingly high binding affinity to the m-opioid
receptor, was found with both (+)- and (ꢀ)-chimo-
nanthine monourethanes (10) and (9). (ꢀ)-Chimo-
nanthine (11), meso-chimonanthine (13) and hodgkinsine
displayed lower affinities, while, as expected, carbo-
methoxychimonanthine (4) did not show affinity for this
receptor. Compared to the trimer hodgkinsine, all the
products showed a fairly minor activity when tested in
the tail-flick model (see Fig. 1); carbomethoxy-
chimonanthine (4), (rac)-chimonanthine and (meso)-
chimonanthine (13) were inactive in this model in the
doses studied, while (ꢀ)-chimonanthine (11) was moder-
ately active. The compound which better resembles the
behaviour of hodgkinsine is (+)-chimonanthine (12).17
The behaviour of the monourethanes (9–10) is particu-
larly intriguing. The derivatives of both (ꢀ)- and (+)-
chimonanthine show the same activity, again supporting
the poor selectivity of the optically active bis-pyrrolidi-
noindoline moiety. Since chimonanthines possess fairly
low binding affinities for the opioid m receptor, the
strong activities shown by 9 and 10 are to be attributed
to the benzylcarbamate moiety. A similar behaviour is
shown by some well known benzamide and benzene-
acetamide amines, which are morphine-like analgesics
with high affinity for the m and k opioid receptors. In
these benzamide and benzeneacetamide amines a pyr-
rolidine type nitrogen is linked through two cyclic
carbon atoms to a tertiary arylamide.20ꢀ22
Among the different chemical structures with affinity for
the opioids receptors, two common features are found:
a basic amino group and an aromatic ring. Effective
receptor interaction is further dependent on the con-
formation assumed by the drug, in which the aromatic
ring and the hydrogen acceptor exhibit a spatial rela-

2138
L. Verotta et al. / Bioorg. Med. Chem. 10 (2002) 2133–2142
Concerning capsaicin-induced pain, only hodgkinsine
and calycosidine presented clear dose-dependent
responses (Fig. 2), hodgkinsine being clearly more
active. The other compounds did not show significant
activity or clear dose–effect relationship.
200 or 40–63 mm) or with neutral Alumina (activity I)
(art 1.0177, Merck). HPLC analyses were carried out on
a Waters 600-MS liquid-liquid chromatograph, con-
nected with a Waters PDA 991 detector and a NEC
386/25 personal computer. A Symmetry C18 column (5
mm, 4.6ꢃ250 mm, Waters) was used, eluted with
MeOH/H2O/Et2NH 80:20:0.1 at a flow rate of 1 mL/
min. Samples were filtered prior to each injection
through Millex FH13 filters (5 mm, Millipore). EI (70 eV)
and CI (isobutane) mass spectra were recorded on a VG
7070 EQ mass spectrometer. 200 and 300 MHz NMR
spectra were recorded on a Bruker AC 200, AC 300 and
Varian Unity Inova 300 spectrometers. Samples were
dissolved in CDCl3 or DMSO-d6 and TMS was used as
reference. DQF-COSY, E-COSY, NOESY, ROESY,
GHSQC, GHMBC experiments were performed by using
the available Varian software (VNMR). Medium pressure
liquid chromatography was carried out with a Buchi 681
Chromatography Pump; a glass column was packed
with Lichroprep RP8 (25–40 mm; art. 9324 Merck).
While it is accepted that the NMDA glutamate receptor
play a prominent role in capsaicin-induced pain, a defi-
nitive conclusion on the presence of a NMDA antago-
nistic property would require NMDA binding data, still
lacking for most of these compounds. Nevertheless,
given that hodgkinsine and psychotridine (a pentameric
pyrrolidinoindoline alkaloid also isolated from P. col-
orata12,13) dose-dependently inhibit capsaicin-induced
pain14 and the binding of the NMDA antagonist
MK801,23 the results obtained with the capsaicin model
can be regarded as an indication of a potential NMDA
antagonism. In any case, only the highest (more than
two units) members of our oligomeric series seem to be
active in this model.
A better correlation between in vivo and opioid in vitro
data is seen with results obtained with tail flick rather
than with capsaicin. This is to be expected, since ther-
mal pain models are known to be more directly asso-
ciated with the opioid system, whereas the glutamate
NMDA receptors are particularly important in capsai-
cin-induced pain. The opioid k subtype receptor does
not seem to be involved in the activity (very low affinity
for all compounds), while the opioid d subtype (not
evaluated) has been reported to be less associated to
both tail flick and capsaicin-induced pain models.24
3-[20(Methoxycarbonyl)amino]ethyl]indole (2). Trypt-
amine (100.3 g, 0.626 mol) was dissolved in dry CH2Cl2
(1.6 L), under nitrogen atmosphere, and dry Et3N (124
mL) was added. The mixture was cooled to 0 ꢄC, and
with stirring, methylchloroformate (55 mL, 0.718 mol)
in dry CH2Cl2 (300 mL) was added dropwise over 30
min. After warming to room temperature, stirring was
continued until the disappearance of tryptamine (2 h 30
min). The mixture was washed with 2% HCl (410 mL),
then with brine (850 mL). The aqueous phase was
extracted twice with CH2Cl2. The pooled organic phases
were dried (Na2SO4) and the solvent evaporated in
vacuum. The residue was crystallized (iPr2O) to give 2
Because NMDA antagonism is of relevance for slowing
opioid tolerance, compounds presenting both opioid
activation and NMDA inhibition properties are of
interest. The biological data so far obtained with these
oligomeric pyrrolinoindoline alkaloids point to the
validity of identifying a synthetic approach for obtain-
ing a compound series in enough quantities for the
necessary comprehensive pharmacological analysis. A
definitive SAR pattern could only be attained if all
possible pyrrolidinoindoline-type oligomers compatible
with the existing stereocenters could be prepared.
(74.3 g, 69%). Mp 79 ꢄC. H NMR (CDCl3) d 8.01 (bs,
1
1H, N1-H), 7.60 (d, J=7.5 Hz, 1H, C7-H), 7.36 (d,
J=7.5 Hz, 1H, C4-H), 7.20 (t, J=7.5 Hz, 1H, C6-H),
7.12 (t, J=7.5 Hz, 1H, C5-H), 7.03 (s, 1H, C2-H), 4.73
(bs, 1H, N30-H), 3.65 (s, 3H,-OCH3), 2.96 (t, J=8 Hz,
2H, C20-H2), 2.52 (m, 2H, C10-H2); EI–MS m/z 218
[M]+ (17), 143 (27), 130 (100). Anal. (C12H14N2O2) C:
calcd 66.04, found 66.10, H calcd 6.47, found 6.40, N
calcd 12.84, found 12.90.
(ꢁ)-Carbomethoxychimonanthine (3) and (meso)-carbo-
methoxychimonanthine (4). The reaction was performed
on compound 2 (45 g), according to ref 16 for 40 h at
room temperature. The crude material was purified on
silica gel eluted with CHCl3/EtOAc 9:1 to give 17% of
starting material and 30% of the mixture 3+4. 13.5 g.
of 3+4 were repeatedly purified on silica gel eluted with
CHCl3/EtOAc 85.15, to give 7.2 g of (ꢁ)-carbo-
methoxychimonanthine (3) and 4.4 g of (meso)-carbo-
methoxychimonanthine (4).
Experimental
Chemistry
Mps have been measured on a Buchi 510 mp apparatus
and are reported uncorrected. Optical rotations have
been measured on a Perkin-Elmer 241 polarimeter. UV
spectra have been recorded on a Hewlett-Packard
8452A spectrophotometer (average concentrations 10ꢀ4
M); CD spectra have been measured on a Jasco J-500
dicrograph (c 10ꢀ4 M). TLC (silica gel 60 F254) were
eluted with CHCl3–MeOH–NH4OH 9:1:0.15. Spots
were revealed by spraying with modified Ehrlich reagent
(p-dimethylaminocinnamaldehyde in 25% methanolic
HCl) or 10% methanolic H2SO4, followed by heating,
or by absorption of the UV light (254 nm). Column
chromatography was performed with silica gel 60 (63–
3. Mp 191 ꢄC (lit 207), Rf 0.31 (CHCl3/EtOAc 8:2). H
1
NMR (CDCl3) d 7.27 (d, J=7.4 Hz, 1H, C4-H), 7.04 (t,
J=7.4 Hz, 1H, C6-H), 6.65 (t, J=7.4 Hz, 1H, C5-H),
6.59 (dd, J=7.4, 3.3 Hz, 1H, C7-H), 6.43 (bs, 1H, N8-
H), 4.73 (s, 1H, C8a-H), 3.53 (s, 3H, OCH3), 3.52 (m,
1H, C2-HB), 2.64 (ddd, J=16.0, 11.0, 5.7 Hz, 1H, C2-
HA), 2.51 (m, 1H, C3-HB), 2.51 (m, 1H, C3-HA); CI MS
m/z 435 [M+H]+ (33); 217 [C12H13N2O2]+ (100), 187

L. Verotta et al. / Bioorg. Med. Chem. 10 (2002) 2133–2142
2139
[C11H11N2O]+ (30), 144 (39), 130 [C9H9N]+ (50), 81
(40), 69 (91). Anal. (C12H14N2O2) C: calcd 66.34, found
66.60, H calcd 6.08, found 5.90, N calcd 12.89, found
13.00.
7. Mp 152–154 ꢄC (iPr2O); [a]D20=ꢀ10.6ꢄ (c 0.34,
CHCl3); Rf 0.29 (toluene/EtOAc 8:2); 1H NMR
(CDCl3) d 8.66 (d, J=5.2 Hz, 1H, ArNHCO), 7.61 (d,
J=8.2 Hz, 1H, C7-H), 7.46 (d, J=7.1 Hz, 2H, Ar2+6-
H2), 7.35 (t, J=7.1 Hz, 2H, Ar3+5-H2), 7.26 (t, J=7.1
Hz, 1H, Ar4-H), 7.18 (m, 1H, C6-H), 6.79 (m, 2H,
C4+5-H2), 5.95 (s, 1H, C8a-H), 5.09 (dq, J=6.9, 5.5
Hz, Ar–CH–N), 3.78 (s, 3H, OCH3), 3.70 (m, 1H, C2-
HA), 2.83 (dd, J=10.1, 9.4 Hz, 1H, C2-HB), 1.87 (m,
2H, C3-HA+B), 1.65 (d, J=7.1 Hz, Ar–CH–CH3); CI
MS m/z: 729 [M+H]+ (97); 625 (10); 582
[C33H35N5O5]+ (57); 435 [C24H27N4O4]+ (12); 366 (38);
279 (24); 262 (18); 219 (78); 217 [C12H13N2O2]+ (100);
187 (35); 174 (16); 159 (24). Anal. (C42H44N6O6) C:
calcd 69.21, found 69.40, H calcd 6.08, found 5.90, N
calcd 11.53, found 11.70.
ꢄ
1
4. Mp 276 C (lit 295) Rf 0.22 (CHCl3/EtOAc 8:2). H
NMR (CDCl3) d 6.97 (t, J=7.9 Hz, 1H, C6-H), 6.57 (d,
J=7.9 Hz, 1H, C4-H), 6.48 (t, J=7.9 Hz, 1H, C5-H),
6.45 (d, J=7.9 Hz, 1H, C7-H), 6.22 (bs, 1H, N8-H),
3.64 (s, 3H, OCH3), 3.64 (m, 1H, C2-HB), 2.80 (m, 1H,
C2-HA), 2.30 (m, 1H, C3-HB), 2.20 (dd, J=12.3, 6.1 Hz,
C3-HA); CI MS m/z 435 [M+H]+ (20), 219 (100), 217
[C12H13N2O2]+ (99), 187 [C11H11N2O]+ (30), 185 (34),
144 (37), 130 [C9H9N]+ (34), 85 (38), 73 (81). Anal.
(C12H14N2O2) C: calcd 66.34, found 66.60, H calcd 6.08,
found 5.90, N calcd 12.89, found 13.10.
(+)-Carbomethoxychimonanthine N8-(ꢀ)-(S)-(ꢀ -me-
thyl-benzyl)carbamate (6) and (ꢀ)-carbomethoxychimo-
nanthine N8-(ꢀ)-(S)-(ꢀ-methyl-benzyl)carbamate (5).
To a stirred solution of (ꢀ)-carbomethoxychimonan-
thine (3) (6.8 g, 15.65 mmol) in dry CHCl3 (125 mL),
kept under nitrogen atmosphere, 30 mL of Et3N were
added, followed by a solution of 5 mL of (ꢀ)-(S)-(a-
methyl-benzyl) isocyanate in dry CHCl3 (20 mL) added
dropwise. The mixture was refluxed for 24 h. The solu-
tion was allowed to cool to room temperature, H2O–
EtOH 1:1 (20 mL), then 5% HCl were added to reach
pH 6 and extracted with CHCl3 (2ꢃ600 mL). The
organic phases were washed with H2O, dried (Na2SO4)
and evaporated to dryness under vacuum. The crude
material (9.1 g) was purified on silica gel (flash chroma-
tography, 8 cm) eluted with CHCl3/EtOAc 6:4, to give
four fractions: fr. A (1.0 g), fr. B (1.3 g, starting mate-
rial, 38%), fr. C (2.8 g) and fr. D (1.5 g).
Fr. C was purified on silica gel eluted with CHCl3/
EtOAc 85:15, to give (ꢀ)-carbomethoxychimonanthine
N8-(ꢀ)-(S)-(a-methyl-benzyl)carbamate (5) (1.68 g,
16.5%). Mp 128–130 ꢄC (iPr2O); [a]2D0=ꢀ261ꢄ (c 0.3,
CHCl3), Rf 0.13 (n-hexane/EtOAc 6:4); 0.167 (CH3OH/
H2O 74:26); tR=8.36 min (CH3OH/H2O/Et2NH
1
80:20:0.1); H NMR (DMSO-d6) d 8.29 (d, J=6.5 Hz,
1H, ArNHCO), 7.56 (d, J=8.3 Hz, 1H, C7-H), 7.44 (d,
J=8.3 Hz, 1H, C4-H), 7.41 (d, J=8.0 Hz, 2H, Ar2+6-
H2), 7.38 (m, 2H, Ar3+5-H2), 7.27 (m, 1H, Ar4-H),
7.24 (t, J=7.5 Hz, 1H, C6-H), 7.17 (d, J=7.5 Hz, 1H,
C40-H), 7.06 (m, 1H, C60-H), 7.02 (t, J=7.5 Hz, 1H, C6-
H), 6.60 (m, 1H, C50-H), 6.58 (d, J=7.0 Hz, 1H, C70-
H), 5.49 (bs, 1H, C8a-H), 4.74 (s, 1H, C80a-H), 4.93 (m,
Ar–CH--N), 3.63 (s, 3H, OCH3), 3.56 (s, 3H, OCH3),
3.70 (m, 1H, C2-HA), 3.51 (m, 2H, C2-HA+B), 2.70 (m,
1H, C2-HB), 2.49 (m, 1H, C3-HA), 2.38 (m, 1H, C3-H
A), 2.22 (m, 1H, C3-HB), 2.16 (m, 1H, C3-HB),1.41 (d,
J=6.5 Hz, Ar–CH–CH3); CI MS m/z: 582 [M+H]+
(58), 478 [Mꢀ106]+ (3), 435 [C24H27N4O4]+ (10), 366
(14), 257 (13), 217 [C12H13N2O2]+ (69), 199 (20), 187
[C11H11N2O]+ (26), 144 (44), 130 [C9H9N]+ (57), 105
(100), 91 [C7H7]+ (84), 81 (50), 71 (92). Anal.
(C33H35N5O5) C: calcd 68.14, found 68.50, H calcd 6.07,
found 5.80, N calcd 12.04, found 12.20.
Fr. A was flash chromatographed on silica gel (petrol/
EtOAc 6:4) obtaining a mixture of the di-carbamates (7)
and (8) (0.64 g) which was crystallized (iPrO2), to give
248 mg of (+)-carbomethoxychimonanthine N8, N80-
(ꢀ)-(S)-(a-methyl-benzyl)dicarbamate (8). The mother
liquors were repeatedly purified on silica gel, eluted with
toluene/EtOAc 85:15, obtaining a fraction (69 mg)
which was crystallized (iPrO2) to give 27 mg of (ꢀ)-car-
bomethoxychimonanthine N8, N80-(ꢀ)-(S)-(a-methyl-
benzyl)dicarbamate (7).
Fr. D was purified on silica gel eluted with petrol/
EtOAc 85:15, to give (+)-carbomethoxychimonanthine
N8-(ꢀ)-(S)-(a-methyl-benzyl)carbamate (6) (1.33 g,
14.6%). Mp 131–133 ꢄC (iPr2O); [a]2D0=+274.4ꢄ (c 0.6,
CHCl3); Rf 0.093 (n-hexane/EtOAc 6:4); 0.167
(CH3OH/H2O 74:26); tR=11.04 min (CH3OH/H2O/
Et2NH 80:20:0.1); 1H NMR (DMSO-d6) d 8.24 (d,
J=7.9 Hz, 1H, ArNHCO), 7.51 (d, J=7.4 Hz, 1H, C4-
H), 7.41 (d, J=7.4 Hz, 1H, C7-H), 7.33 (m, 2H,
Ar2+6-H2), 7.33 (m, 2H, Ar3+5-H2), 7.32 (d, J=7.0
Hz, 1H, C40-H), 7.24 (m, 1H, Ar4-H), 7.23 (m, 1H, C6-
H), 7.11 (t, J=7.6 Hz, 1H, C60-H), 7.04 (t, J=7.3 Hz,
1H, C6-H), 6.70 (t, J=7.0 Hz, 1H, C50-H), 6.66 (d,
J=7.0 Hz, 1H, C70-H), 5.27 (bs, 1H, C8a-H), 4.54 (s,
1H, C80a-H), 4.90 (m, Ar–CH–N), 3.61 (s, 3H, OCH3),
3.55 (s, 3H, OCH3), 3.69 (m, 1H, C2-HA), 3.60 (m, 1H,
C20-HA), 2.70 (m, 2H, C2-HB+C20-HB), 2.54 (m, 1H,
C3-HA), 2.49 (m, 1H, C30-HA), 2.34 (m, 1H, C3-HB),
2.31 (m, 1H, C30-HB), 1.41 (d, J=7.0 Hz, Ar–CH–CH3);
CI MS m/z: 582 [M+H]+ (58), 522 [Mꢀ59]+ (2), 478
8. Mp 262 ꢄC (iPr2O); [a]D20=+160.2ꢄ (c 0.9, CHCl3); Rf
1
0.33 (toluene/EtOAc 8:2); H NMR (CDCl3) d 8.37 (d,
J=7.6 Hz, 1H, ArNHCO), 7.81 (d, J=8.2 Hz, 1H, C7-
H), 7.46 (d, J=7.7 Hz, 2H, Ar2+6-H2), 7.36 (t, J=7.3
Hz, 2H, Ar3+5-H2), 7.27 (m, 1H, C6-H), 7.26 (m, 1H,
Ar4-H), 7.15 (d, J=7.7 Hz, 1H, C4-H), 7.02 (t, J=7.3
Hz, C5-H), 5.69 (bs, 1H, C8a-H), 5.07 (dq, J=7.2 Hz,
Ar–CH–N), 3.74 (s, 3H, OCH3), 3.87 (dd, J=10.4, 6.5
Hz, 1H, C2-HA), 2.87 (m, 1H, C2-HB), 2.16 (m, 2H, C3-
HA+B), 1.61 (d, J=7.0 Hz, Ar–CH–CH3); CI MS m/z:
729 [M+H]+ (100), 625 (12), 582 [C33H35N5O5]+ (57),
435 [C24H27N4O4]+ (12), 366 (39), 279 (21), 262 (20),
257 (19), 219 (75), 217 [C12H13N2O2]+ (97), 187 (29),
174 (15), 159 (23). Anal. (C42H44N6O6) C: calcd 69.21,
found 69.40, H calcd 6.08, found 5.90, N calcd 11.53,
found 11.70.

2140
L. Verotta et al. / Bioorg. Med. Chem. 10 (2002) 2133–2142
[Mꢀ106]+ (5.4), 435 [C24H27N4O4]+ (9.2), 366 (17), 317
[C10H21N4]+ (3), 217 [C12H13N2O2]+ (92), 187
[C11H11N2O]+ (24), 144 (31), 130 [C9H9N]+ (55), 105
(100). Anal. (C33H35N5O5) C: calcd 68.14, found 68.40,
H calcd 6.07, found 5.80, N calcd 12.04, found 12.20.
346 M+(2), 174 (43), 173 (94), 172 (100), 171 (51), 157
(14), 143 (16), 131 (68), 130 [C9H9]+ (100), 117 (16), 103
(19), 77 (25). Anal. (C22H26N4) C: calcd 76.27, found
76.30, H calcd 7.56, found 7.40, N calcd 16.17, found
16.30.
(ꢀ)-chimonanthine N8-(ꢀ)-(S)-(ꢀ-methyl-benzyl)carba-
mate (9). To a suspension of LiAlH4 (890 mg, 24.1
mmol) in dry THF (50 mL), 5 (1.3 g, 2.24 mmol, in 70
mL of dry THF), was added dropwise at 0 ꢄC. The
reaction is warmed to reflux for 3 h 30 min. The mixture
was left to reach room temperature, then was carefully
basified to pH 9 with 10% NaOH and filtered on Celite
which was repeatedly washed with THF. The solution
was concentrated to water and extracted with CHCl3
(2ꢃ250 mL). The organic phases were washed with
H2O, dried (Na2SO4) and evaporated to dryness under
vacuum.
(+)-Chimonanthine N8-(ꢀ)-(S)-(ꢀ-methyl-benzyl)carba-
mate (10). According to the procedure used for making
compound 9, from compound 6, (+)-chimonanthine
N8-(ꢀ)-(S)-(a-methyl-benzyl)carbamate (10) (605 mg,
59%) and (+)-chimonanthine (12) (120 mg, 16%) were
obtained.
10. Mp=106–108 ꢄC (n-hexane/iPr2O 1:1); [a]D20=
+119.7ꢄ (c 0.8, CHCl3); Rf 0.37 (EtOAc/Et2NH 96:4)
1H NMR (DMSO-d6) d 7.06 (m, 1H, ArNHCO), 7.50
(d, J=7.8 Hz, 1H, C7-H), 7.44 (d, J=7.4 Hz, 2H,
Ar2+6-H2), 7.35 (t, J=7.2 Hz, 2H, Ar3+5-H2), 7.26 (t,
J=7.2 Hz, 1H, Ar4-H), 7.18 (d, J=7.6 Hz, 1H, C4-H),
6.96 (t, J=7.9 Hz, 1H, C6-H), 6.87 (d, J=6.5 Hz, 1H,
C40-H), 6.75 (t, J=7.4 Hz, 1H, C5-H), 6.73 (t, J=7.4
Hz, 1H, C60-H), 6.27 (d, J=7.4 Hz, 1H, C70-H), 6.07
(bs, 1H, N80-H), 5.45 (m, 2H, C8a+80a,-H2), 5.06 (dq,
J=7.2 Hz, 1H, Ar–CH–N), 2.62 (m, 1H, C2-HA), 2.53
(m, 1H, C20-HA), 2.41 (m, 1H, C3-HA); 2.33 (s, 3H,
N10-CH3), 2.23 (m, 1H, C2-HB), 2.28 (m, 1H, C20-HB),
2.28 (s, 3H, N1-CH3), 1.98 (m, 1H, C20-HA), 1.92 (1H,
m, C3-HB), 1.52 (s, 3H, Ar–CH–CH3); CI MS m/z: 494
[M+H]+ (77), 376 [M+H-2ꢃCH3OCO]+ (13), 349
(17), 347 [M+H-147]+ (16), 215 (14), 186 (16), 174
[C11H14N2]+ (99), 172 [C11H12N2]+ (100), 158 (17),
144 (30), 132 (43), 130 [C9H9N]+ (47), 105 (48), 91
[C7H7]+ (15). Anal. (C31H34N5O) C: calcd 75.43, found
75.60, H calcd 7.15, found 6.90, N calcd 14.19, found
14.00.
The crude material was purified on silica gel, eluted with
EtOAc/Et2NH 98:2 (800 mL) and 96:4 (1 L), to give
(ꢀ)-chimonanthine N8-(ꢀ)-(S)-(a-methyl-benzyl)carba-
mate (9) (750 mg, 68%) and 270 mg of a fraction which
was further purified on silica (EtOAc/Et2NH 97:3) to
give (ꢀ)-chimonanthine (11) (145 mg, 18%).
9. Mp 112–113 ꢄC (n-hexane/iPr2O 1:1); [a]2D0=ꢀ83.4ꢄ (c
0.7, CHCl3); Rf 0.24 (EtOAc/Et2NH 96:4) 1H NMR
(DMSO-d6) d 8.32 (s, 1H, ArNHCO), 7.52 (d, J=8.0
Hz, 1H, C7-H), 7.46 (d, J=7.4 Hz, 2H, Ar2+6-H2),
7.36 (t, J=7.1 Hz, 2H, Ar3+5-H2), 7.25 (t, J=7.1 Hz,
1H, Ar4-H), 7.16 (d, J=7.5 Hz, 1H, C4-H), 6.86 (m,
1H, C6-H), 6.84 (m, 1H, C40-H), 6.68 (1H, C5-H), 6.66
(m, 1H, C60-H), 6.24 (d, J=7.2 Hz, 1H, C70-H), 6.06
(bs, 1H, N80-H), 5.62 (m, 2H, C8a+80a,-H2), 5.02 (q,
J=7.2 Hz, 1H, Ar–CH–N), 2.70 (m, 1H, C2-HA), 2.60
(m, 1H, C20-HA), 2.50 (m, 1H, C3-HB), 2.44 (s, 3H, N10-
CH3), 2.41 (m, 1H, C30-HA), 2.32 (s, 3H, N1-CH3), 2.30
(m, 1H, C20-HB), 2.25 (m, 1H, C2-HB), 2.01 (m, 1H, C3-
HA)1.95 (m, 1H, C30-HB), 1.52 (s, 3H, Ar–CH–CH3); CI
MS m/z: 494 [M+H]+ (32), 347 [M+H-147]+ (6), 319
[C20H21N3O]+ (37), 174 [C11H14N2]+ (75), 172
[C11H12N2]+ (71), 131 (37), 130 [C9H9N]+ (42), 108
(55), 105 (62), 91 [C7H7]+ (100). Anal. (C31H34N5O) C:
calcd 75.43, found 75.60, H calcd 7.15, found 6.80, N
calcd 14.19, found 14.00.
12. Mp 168–170 ꢄC (iPr2O/CHCl3 3:1); [a]2D0=+270ꢄ (c
0.9, EtOH); Rf 0.32 (CHCl3/CH3OH/NH3 9:1:0.15)
tR=6.16 min (CH3OH/H2O/Et2NH 80:20:0.1); 1H
NMR, 13C NMR and MS identical to 11. Anal.
(C22H26N4) C: calcd 76.27, found 76.40, H calcd 7.56,
found 7.40, N calcd 16.17, found 16.20.
(ꢀ)-Chimonanthine (11). Compound 9 (27.5 mg, 55.7
mmol) was treated with 1 N sodium pentoxide (1.8 mL)
and the mixture was refluxed for 3 h. At room tem-
perature, 0.5 N HCl was added to reach pH 8; the mix-
ture was diluted with H2O and extracted with CHCl3
(2ꢃ15 mL). The organic phases were washed with
water, dried (Na2SO4) and evaporated to dryness under
vacuum. The crude material was purified on silica gel,
eluted with EtOAc/Et2NH 96:4, to give (ꢀ)-11 (14.1 mg,
73%). The same procedure was followed to obtain 12
from 10 (yield 75%).
11. Mp 171–172 ꢄC (iPr2O/CHCl3); [a]2D0=ꢀ310ꢄ (c 0.8,
EtOH); Rf 0.32 (CHCl3/CH3OH/NH3 9:1:0.15) tR=6.16
min (CH3OH/H2O/Et2NH 80:20:0.1); 1H NMR
(CDCl3) d 7.23 (d, J=7.5 Hz, 1H, C4-H), 7.04 (t, J=7.5
Hz, 1H, C6-H), 6.71 (t, J=7.5 Hz, 1H, C5-H), 6.59 (d,
J=7.5 Hz, 1H, C7-H), 4.48 (bs, 1H, C8a-H), 2.59 (m,
2H, C2-HA+B), 2.58 (m, 1H, C3-HA), 2.37 (s, 3H, N–
1
CH3), 2.12 (dt, J=12.0, 6.4 Hz, 1H, C3-HB), H NMR
(DMSO-d6) d 7.04 (bs, 1H, C4-H), 6.81 (bs, 1H, C6-H),
6.40 (bs, 1H, C5-H), 6.37 (bs, 1H, C7-H), 6.11 (bs, 1H,
NH), 4.11 (bs, 1H, C8a-H), 2.59 (m, 1H, C2-HA), 2.46
(m, 1H, C3-HA), 2.31 (m, 1H, C2-HB), 2.26 (s, 3H, N–
CH3), 1.85 (m, 1H, C3-HB); 13C NMR (CDCl3) d 150.8
(s, C7a), 133.2 (s, C4a), 128.5 (d, C6), 124.9 (d, C4),
119.1 (d, C5), 109.8 (d, C7), 85.6 (s, C8a), 63.7 (s, C3a),
53.0 (t, C2), 37.4 (q, N–CH3), 35.9 (t, C3); EI MS m/z:
(meso)-Chimonanthine (13). According to the procedure
used for making 9 and 10, compound 4 was treated with
LiAlH4 in THF to give 13 (yield 45%). Mp 194–196 ꢄC
(lit 198, 201 ꢄC) (EtOH); Rf=0.25 (CHCl3/CH3OH/
NH3 9:1:0.15) tR=10.20 min (CH3OH/H2O/Et2NH
80:20:0.1); 1H NMR (CDCl3) d 7.06 (m, 2H, C6-
H+C5-H), 6.60 (d, J=8.0 Hz, 2H, C4-H+C7-H), 2.86
(m, 1H, C2-HA), 2.52 (m, 1H, C2-HB), 2.50 (m, 1H,

L. Verotta et al. / Bioorg. Med. Chem. 10 (2002) 2133–2142
2141
1
C3-HA), 2.45 (s, 3H, N-CH3), 2.11 (m, 1H, C3-HB), H
NMR (DMSO-d6) d 6.88 (m, 2H, C6-H+C5-H), 6.30
(d, J=8.0 Hz, 2H, C4-H+C7-H), 2.73 (m, 1H, C2-HA),
2.48 (m, 1H, C3-HA), 2.30 (s, 3H, N–CH3), 2.23 (m, 1H,
C2-HB), 1.88 (m, 1H, C3-HB); 13C NMR (CDCl3) d
152.5 (s, C7a), 133.7 (s, C4a), 128.9 (d, C6), 125.2 (d,
C4), 119.1 (d, C5), 109.5 (d, C7), 84.1 (s, C8a), 64.7 (s,
C3a), 53.1 (t, C2), 37.5 (t, C3), 36.3 (q, N–CH3); 13C
NMR (DMSO-d6) d 153.5 (s, C7a), 133.6 (s, C4a), 128.4
(d, C6), 124.7 (d, C4), 117.0 (d, C5), 108.0 (d, C7), 83.6
(s, C8a), 63.8 (s, C3a), 52.3 (t, C2), 36.3 (q, N–CH3),
31.6 (t, C3); EI MS m/z: 346 M+ (13), 245 (12), 174 (88),
173 (89), 172 (100), 171 (75), 157 (16), 143 (17), 131 (35),
130 [C9H9]+ (82), 117 (15), 103 (11). Anal. (C22H26N4)
C: calcd 76.27, found 76.30, H calcd 7.56, found 7.50, N
calcd 16.17, found 16.15.
Binding experiments were performed in 25 mM mono-
basic potassium phosphate buffer, containing 3 mM
MgCl2, pH 7.4 in polypropylene 96-deep-well plates.
Incubation was carried on for 60 min at 25 ꢄC at the
final volume of 0.5 mL (k assay) or 0.7 mL (m assay).
The reaction was terminated by filtration using a Pack-
ard Filtermate harvester containing a GF/B Unifilter
plate. After filtration, Unifilter plates were dried, each
well filled with 50 mL of Packard Microscint 30 and
radioactivity counted by a Packard Topcount NXT.
IC50 values were determined using the non linear least-
squares fitting program GraFit (Erithacus Software
Ltd, Horley, UK)27 and were then trasformed into Ki
values by applying the Cheng and Prusoff equation.28
Pharmacology
Calycosidine. Hodgkinsine ([a]2D0=ꢀ33.6) (94 mg) was
treated with 1 N CH3COOH (2 mL). After 2 h at rt, the
solution was refluxed for 2 h. At the formation of by-
products, the reaction was poured in ice, diluted to
Animals. Male albino mice, CF1, 30–35 g, maintained at
20ꢁ2 ꢄC, 12 h light/dark cycle, with food and water ad
libitum were used in all experiments.
pH
9 with 15% NH4OH, then extracted with
CH2Cl2. After drying, the mixture was taken to dry-
ness under vacuum and purified by flash chromato-
graphy (Ø 1.7 cm, EtOAc–Et2NH 98:2, 60 mL).
Combined fractions give 52 mg of calycosidine.
Drugs. Morphine sulfate, naloxone, capsaicin, MK-801
(dizolcipine) were acquired from Sigma (USA). With
the exception of mesocarboxychimonanthine diluted in
Tween (80), all other compounds were transformed into
salts by adding stechiometric amounts of HCl and dilu-
ted in distilled water.
20
[a]D =ꢀ22ꢄ (c 0.1, CHCl3); H NMR (CDCl3) d 7.12
(dd, J=7.4, 1.0 Hz, 1H, C400-H), 7.01 (dd, J=7.8, 1.4
Hz, 1H, C4-H), 6.90 (d, J=6.9 Hz, 1H, C60-H), 6.86 (t,
J=7.6 Hz, 1H, C6-H), 6.82 (t, J=7.7 Hz, 1H, C600-H),
6.69 (d, J=7.0 Hz, 1H, C40-H), 6.56 (t, J=7.0 Hz, 1H,
C5-H), 6.55 (t, J=6.9 Hz, 1H, C500-H), 6.49 (t, J=6.9
Hz, 1H, C50-H), 6.45 (d, J=7.6 Hz, 1H, C7-H), 6.42 (d,
J=7.7 Hz, 1H, C700-H), 5.84 (s, 1H, C800a-H), 5.02 (d,
J=3.6 Hz, 2H, NH), 4.96 (bs, 1H, NH), 4.50 (s, 1H,
C80a-H), 4.09 (d, J=3.9 Hz, 1H, C8a-H), 2.57 (m,
1
Tail-flick test. Analgesia was assessed with a Tail flick
apparatus (Albarsch Electronic Equipment) following
the method detailed elsewhere.9 Forty-five minutes
before testing, animals were placed individually in
acrylic chambers (20ꢃ20ꢃ20 cm) which also served for
observation. Pre-drug latency (reaction time for remov-
ing the tail from heat source) was obtained as the mean
of three measures (after each measure animals were
returned to the observation chambers for 2 min). At this
point, animals presenting two measures of 6 (or more)
seconds were discarded. A cut-off time of 10 s was used
to prevent tissue damage. Treatments were administered
(ip) immediately after the third pre-drug measure.
Thirty min later, another set of three measures was
taken and the mean considered as post-drug reaction
time. Reversibility by naloxone (ip, 10.0 mg/kg) was
tested by administering naloxone 10 min before treat-
ments. Statistical analysis used Kruskal–Wallis/Mann–
Whitney. Differences in pre- and post-drug latencies
were analyzed by the Wilcoxon test. Data are presented
as% of Maximum Possible Effect (% MPE), obtained
through the following formula:% MPE=T1ꢀT0/
T2ꢀT0ꢃ100 where, T1=time post-drug, T0=time pre-
drug and T2=cut-off time (10 or 20 s, respectively).
Statistical analysis used ANOVA/Student–Newmann–
Keuls. Differences in pre- and post-drug latencies were
analyzed by the Wilcoxon test.
0
2H, C200-HA+B), 2.31 (s, 3H, CH3 ), 2.30 (m, 1H,
C300-HA), 2.28 (s, 3H, CH3’), 2.27 (m, 1H, C20-HA),
2.18 (m, 1H, C300-HB), 2.09 (s, 3H, CH3), 2.09 (m, 1H,
C20-HB), 2.08 (m, 1H, C30-HB), 2.01 (m, 2H, C2-HA+B),
1.75 (m, 1H, C3-HB), 1.12 (m, 1H, C30-HA), 0.86 (m,
1H, C3-HA).
Opioid binding assay
Binding experiments have been performed in mem-
branes prepared from Chinese hamster ovary (CHO) or
human embrionic kidney (HEK) cells stably expressing
human cloned m- and k-opioid receptors, respectively.
A stable expression of m receptors (h-MOR) in CHO cell
line has been performed in house, using pCDN vec-
tors.25 Membranes were prepared by lysis in hypotonic
phosphate-buffer according to the method described by
Scheideler and R. S. Zukin.26 The highly potent and
selective radioligands [3H]-[d-Ala,2 Mephe,4 Gly-ol5]en-
kephalin ([3H]-DAMGO, 50 Ci/mmol, New England
Nuclear, Bruxelles, Belgium), and [3H]-U-69593 (63 Ci/
mmol, Amersham, Italy) were used to label m- and k-
opioid receptors, respectively.25
Capsaicin-induced pain. Experiments were performed
according to a previously described method:29,30 20 min
before the experiment, animals were placed individually
in acrylic boxes, which also serve as observation cham-
Non-specific binding was determined in the presence of
10 mM Naloxone.

2142
L. Verotta et al. / Bioorg. Med. Chem. 10 (2002) 2133–2142
bers. After this adaptation period, 20 mL of capsaicin
(1.6 mg/paw) was injected under the dorsal skin of the
right hindpaw using a Hamilton microsyringe with a 26-
gauge needle. Treatments were administered sc (neck) or
ip 30 min before capsaicin and animals were individu-
ally observed for 5 min after capsaicin administration.
The time spent in licking the injected paw was recorded
and taken as the pain index. Data were analyzed
through ANOVA/Student–Newmann–Keuls.
15. The enantioselective approaches towards the synthesis of
the three chimonanthines are reported in: (a) Hoyt, S.B.;
Overman, L.H. Org. Lett. 2000, 2, 3241. (b) Overman, L.E.;
Paone, D.V.; Stearns, B.A. J. Am. Chem. Soc. 1999, 121, 7702.
(c) Overman, L.E.; Larrow, J.F.; Stearns, B.A.; Vance, J.M.
Angew. Chem. Int. Ed. 2000, 39, 213.
16. Nagakawa, M.; Sugumi, H.; Kodato, S.; Hino, T. Tetra-
hedron Lett. 1981, 22, 5323.
17. It has been reported that both the two enantiomers of
eseroline show comparable affinity with the opiate receptors,
with the (ꢀ)-enantiomer being twice as active as the (+)-
enantiomer.18 In vivo assays have demonstrated that only (ꢀ)-
eseroline has potency comparable to morphine.18 In our case,
(ꢀ)-chimonanthine shows stronger affinity to m receptors than
the (+)-enantiomer, while opposite results are obtained in the
tail-flick test. Nevertheless, a direct comparison with eseroline
is risky, since the phenol OH in eseroline can play a crucial
role, acting as hydrogen donor in the formation of hydrogen
bondings with the receptor. Moreover, eseroline closely
resembles the morphinane structures. Very recently, a paper
has appeared19 describing the strong affinity and selectivity of
3-deoxy, 14 b-cinnamoylamino derivatives of morphinone
(deoxyclocinnamox). Accordingly, in this paper, the authors
report that the cinnamoylamino group appears to have a
grater influence on opioid binding than the presence of a phe-
nolic hydroxyl group.
18. Schonenberger, B.; Jacobson, A. E.; Brossi, A.; Streaty,
R.; Klee, W. A.; Flippen-Anderson, J. L.; Gilardi, R. J. Med.
Chem. 1986, 29, 2268.
19. Derrick, I.; Neilan, C. L.; Andes, J.; Husbands, S. M.;
Woods, J. H.; Traynor, J. R.; Lewis, J. W. J. Med. Chem.
2000, 43, 3348.
20. de Costa, B. R.; Bowen, W. D.; Hellewell, S. B.; George,
C.; Rothman, R. B.; Reid, A. A.; Walker, J. M.; Jacobson,
A. E.; Rice, K. C. J. Med. Chem. 1989, 32, 1996.
21. Lucet, D.; Le Gall, T.; Mioskowsky, C. Angew. Chem.,
Int. Ed. 1998, 37, 2580.
Acknowledgements
This work was supported by CNR and MURST of
Italy, and CNPq and FAPERGS of Brazil. The authors
are grateful to Loredana Antonini for her excellent
technical assistance.
References and Notes
1. Mattison, N.; Trimble, A. G.; Lasagna, L. Clin. Pharmacol.
and Ther. 1988, 43, 290.
2. Olson, G. A.; Olson, R. D.; Kastin, A. J. Peptides 1993, 14,
1339.
3. Wollemann, S. B.; Simon, J. Life Sci. 1992, 52, 599.
4. Reid, W.V.; Laird, S.A.; Gamez, R.; Sittenfeld, A.; Janzen,
D.H.; Gollin, M.A.; Juma, C.A. In Biodiversity Prospecting:
Using Genetic Resources for Sustainable Development; Reid,
W.V., Laird, S.A., Eds.; World Resources Institute: Baltimore,
1993; p 1.
5. Balandrin, M. F.; Klocke, J. A.; Wurtele, E. S.; Bollinger,
W. H. Science 1985, 228, 1154.
6. Tyler, V. E. Econ. Bot. 1986, 40, 93.
7. Cunningham, A.B. In Ethics, Ethnobiological Research and
Biodiversity; Lindsay, B., Ed.; WWF International: Gland,
Switzerland; 1993: p 44.
22. Cheney, B. V.; Szmuszkovic, J.; Lahti, R. A.; Zichi, D. A.
J. Med. Chem. 1985, 28, 1853.
23. Amador, T. A.; Verotta, L.; Nunes, D. S.; Elisabetky, E.
Phytomedicine 2001, 8, 202.
8. Elisabetsky, E.; Castilhos, Z. C. Int. J. Crude Drug Res.
1990, 28, 309.
24. Dondio, G.; Ronzoni, S.; Petrillo, P. Exp. Opin. Ther. Pat.
1999, 9, 353.
25. Kotzer, C. J.; Hay, D. W. P.; Dondio, M.; Giardina,
G. A. M.; Petrillo, P.; Underwood, D. J. Pharmacol. Exp.
Ther. 2000, 292, 803.
9. Elisabetsky, E.; Amador, T. A.; Albuquerque, R. R.;
Nunes, D. S.; Carvalho, A. C. T. J. Ethnopharmacol. 1995, 48,
77.
10. Amador, T. A.; Elisabetsky, E.; Souza, D. O. Neurochem.
Res. 1996, 21, 97.
26. Scheideler, M. A.; Zukin, R. S. J. Biol. Chem. 1990, 265,
15176.
27. Leatherbarrow, R. J. Trends Biochem. Sci. 1990, 15, 455.
28. Cheng, Y.-L.; Prusoff, W. H. Biochem. Pharmacol. 1973,
22, 3099.
29. Correˆ a, C. R.; Kyle, D. J.; Chakravarty, S.; Calixto, J. B.
Brit. J. Pharmacol. 1996, 110, 552.
30. Sakaruda, T.; Katsumata, K.; Tan-No, K.; Sakurada, S.;
Kisara, K. Neuropharmacology 1992, 31, 1279.
11. Elisabetsky, E.; Amador, T. A.; Leal, M. B.; Nunes,
D. S.; Carvalho, A. C. T.; Verotta, L. Cieˆncia Cultura 1997,
49, 378.
12. Verotta, L.; Pilati, T.; Tato, M.; Elisabetsky, E.; Amador,
`
T. A.; Nunes, D. S. J. Nat. Prod. 1998, 61, 392.
13. Verotta, L.; Peterlongo, F.; Elisabetsky, E.; Amador,
T. A.; Nunes, D. S. J. Chromatogr. A 1999, 841, 165.
14. Amador, T. A.; Verotta, L.; Nunes, D. S.; Elisabetsky, E.
Planta Med. 2000, 66, 1.